Studies will continue on the mechanism of energy coupling to ATP synthesis in two prokaryotic systems in which the energetics of oxidative phosphorylation diverge significantly from the predictions of the chemiosmotic model. That model provides a broadly applicable description, currently the dominant one, for energy-coupling to bioenergetic work. Were there to be organelles or organisms that transduce respiration-derived energy to ATP synthesis in a nonchemiosmotic mode, then the pharmacology, and potentially the genetics, of this central aerobic process would be different than in a totally chemiosmotic mode. The chemiosmotic model posits that ATP synthesis during oxidative phosphorylation is completely coupled to an electrochemical ion gradient, be it formed during respiration or imposed artificially. By contrast, both extremely alkaliphilic Bacillus firmus OF4 and certain uncoupler-resistant mutant strains of Bacillus subtilis exhibit maximal ATP synthesis at sub-maximal levels of the bulk respiration-derived force, and not in response to larger artificially imposed gradients. A working hypothesis that accounts for all the observations in these systems will be explicitly tested, and aspects of possible alternative explanations will continue to be explored. Four specific aims are described. (1) Proposed pK-regulated closure of the F-O of the alkaliphile ATP synthase to bulk protons will be examined. Such gating would create a kinetic barrier to loss of protons that might reach the F-O through intramembrane transfers, and would account for the failure of imposed potentials to energize synthesis at pH>9.5. Proton flux experiments will be conducted in mutants with leaky synthases and in F1F- O-proteoliposomes at various pH values. Also, a possible shift in the titration-like energization behavior of the synthase will be examined upon deuteration. (2) Protein-protein interactions between the caa3-type terminal oxidase and the ATP synthase of the alkaliphile, that could allow direct intramembrane proton transfer, will be probed. Approaches will include studies of thermotropic behavior of singly and co-reconstituted preparations of the putative interacting partners, effects of the putative interacting partner on the labelling pattern of the F-O c-subunit by 3- (trifluoromethyl-3-m[125]phenyl)diazirine, and cross-linking experiments. Analogous clustering of ATP synthase and respiratory chain elements in the uncoupler-resistant mutants will be examined. (3) Gating and interactive properties will also be examined in energy-coupling mutants of the alkaliphile with alterations in the synthase, including those developed by directed changes in interesting "alkaliphile-specific" motifs in the F-O. (4) As part of examinations of possible alternatives to the central working hypothesis, e.g. variable coupling stoichiometry or surface trapping of protons, direct determinations of the H+/ATP stoichiometry of the alkaliphile ATP synthase will be made as a function of pH, and the pH at the outer surface of the coupling membrane will be determined.